[Subchromosomal microdeletion identified by molecular karyotyping using DNA microarrays (array CGH) in Rett syndrome girls negative for MECP2 gene mutations].

Molecular karyotyping using DNA microarrays (array CGH) was applied for identification of subchromosomal microdeletions in a cohort of 12 girls with clinical features of RETT syndrome, but negative for MECP2 gene mutations. Recurrent microdeletions of MECP2 gene in chromosome X (locus Xq28) were identified in 5 girls of 12 studied. Probably RTT girls with subchromosomic microdeletions in Xq28 could represent a special subtype of the disease, which appears as clinically milder than the classic form of disease. In one case, an atypical form of RTT was associated with genomic abnormalities affecting CDKL5 gene and region critical for microdeletion Prader-Willi and Angelman syndromes (15q11.2). In addition, data are presented for the first time that genetic variation in regions 3p13, 3q27.1, and 1q21.1-1q21.2 could associate with RTT-like clinical manifestations. Without application of molecular karyotyping technology and bioinformatic method of assessing the pathogenic significance of genomic rearrangements these RTT-like girls negative for MECP2 gene mutations were considered as cases of idiopathic mental retardation associated with autism. It should be noted that absence of intragenic mutations in MECP2 gene is not sufficient criteria to reject the clinical diagnosis of RTT. To avoid errors in the genetic diagnosis of this genetically heterogeneous brain disease molecular cytogenetic studies using high resolution oligonucleotide array CGH (molecular karyotyping) are needed.

[Genomic abnormalities in children with mental retardation and autism: the use of comparative genomic hybridization in situ (HRCGH) and molecular karyotyping with DNA-microchips (array CGH)].

Genomic abnormalities occur with high frequency in children with mental retardation and autistic spectrum disorders (ADS). Molecular karyotyping using DNA microarrays is a new technology for diagnosis of genomic and chromosomal abnormalities in autism implemented in the fields of biological psychiatry and medical genetics. We carried out a comparative analysis of the frequency and spectrum of genome abnormalities in children with mental retardation and autism of unknown etiology using high-resolution comparative genomic methods for hybridization (HRCGH) and molecular karyotyping (array CGH). In a study of 100 children with autism, learning difficulties and congenital malformations by HRCGH, we identified genomic rearrangements in 46% of cases. Using array CGH we examined 50 children with autism. In 44 cases out of 50 (88%), different genomic abnormalities and genomic variations (CNV - copy number variations) were identified. Unbalanced genomic rearrangements, including deletions and duplications, were found in 23 cases out of 44 (52%). These data suggest that genomic abnormalities which are not detectable by common methods of chromosome analysis are often discovered by molecular cytogenetic techniques in children autism spectrum disorders. In addition, 54 children with idiopathic mental retardation and congenital malformations (31 boys and 23 girls) without autism spectrum disorders were examined using molecular karyotyping and microarray containing an increased number of DNA samples for genomic loci of chromosome X. Deletions and duplications affecting different regions of the chromosome X were detected in 11 out of 54 children (20.4%).

[Genomic instability in the brain: etiology, pathogenesis and new biological markers of psychiatric disorders].

The latest advances in molecular medicine, medical genetics and neurobiology have provided for a new look at processes occurring in cells of the brain and have allowed to discover previously unknown phenomena associated with mental traits and to propose new biomedical direction which include genomics, psychiatry and neurobiology - brain genomics. The application of modern molecular and cellular technologies of genome analysis in the brain in common psychiatric disorders (autism, schizophrenia and Alzheimer's disease) has shown that genomic instability is a phathogenetic mechanism of central nervous system abnormalities and plays a role in the brain development. Genomic disbalance alters neural homeostasis leads to cell death and is an important biological marker of psychiatric disorders which determine genomic pathways. These alterations lead to synaptic disfunction and neurodegeneration. In the present review, the main advances of brain genomics and potential application in diagnostic, clinical and therapeutic practice.

[Original molecular cytogenetic approach to determining spontaneous chromosomal mutations in the interphase cells to evaluate the mutagenic activity of environmental factors].

Standard cytogenetic methods are presently used to analyze chromosome anomalies in human somatic cells in order to evaluate the mutagenic activity of environmental factors. Their application is associated with the fact that uncultured cells cannot be analyzed. In this connection there is an urgent need to devise and introduce studies of the number and structure of interphase chromosomes. The paper describes the molecular cytogenetic approach to determining spontaneous chromosomal mutations in the interphase cells to evaluate the mutagenic activity of environmental factors, which is based on the original methods of interphase fluorescence in situ hybridization (FISH) and multicolor chromosome staining and on quantitative FISH and immune FISH. The proposed set of the methods has been tested examining 400000, 200000, and 1600000 embryonic, extraembryonic, and postnatal tissue cells, respectively. The analysis has yielded data on sporadic chromosomal mutations and shown that the proposed complex of the methods can be successfully used to evaluate the mutagenic activity of environmental factors leading to sporadic chromosomal mutations.

[Molecular cytogenetic methods for studying interphase chromosomes in human brain cells].

One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.

[Identification of candidate genes of autism on the basis of molecular cytogenetic and in silico studies of the genome organization of chromosomal regions involved in unbalanced rearrangements].

Autism is one of the most widely spread mental diseases among children. Different genetic anomalies make a considerable contribution to the etiology of this disease; therefore, the identification of candidate genes of autism can be regarded as a topical task of modern medical genetics. The molecular cytogenetic examination of children with autism was carried out using high-resolution comparative genome hybridization and subsequent in silico analysis of chromosomal regions involved in unbalanced rearrangements. Five of 126 (4%) children with autism had unbalanced rearrangements of chromosomes 5, 17, 21 (deletions) and chromosomes 4 and 22 (duplications). The following candidate genes were identified in children with autism by in silico analysis: SCARB2, TPPP, PDCD6, SEPT5, GP1BB, PI4KA, NPTX1, STCH, NRIP1, and CXADR. These methods also allowed us to find a possible association between gene clusterization and the formation of the described chromosomal rearrangements. Thus, this study demonstrates that the molecular cytogenetic and bioinformatic methods can be successfully used to search for candidate genes of different diseases and analyze the genome organization.

[Instability of chromosomes in human nerve cells (normal and with neuromental diseases)].

It is assumed that the genetic mechanism of pathogenesis of such widely spread neural and mental diseases as schizophrenia (SZ), autism, ataxia-telangiectasia (AT), and Alzheimer's disease (AD) is associated with structural and functional genomi? instaility in brain cells. Aneuploidy is one of the most important biological markers of genomic instability. The currently available methods of molecular cytogenetics (I-mFISH, QFISH, and ICS-MCB) facilitate the solution of numerous fundamental biological problems, including analysis ofgenomic variations in brain cells. Using these methods, we have studied for the first time aneuploidy in human embryo and adult brain cells (normal and with AT, AD, and SZ) as well as in blood cells of children with autism. The level of aneuploidy was increased two- to threefold in the embryo brain with a subsequent reduction of the number of abnormal cells in the adult brain. In the case of SZ, mosaic aneuploidy for chromosomes 1, 18, and X was found. The study of blood cells from children with autism showed chromosomal mosaicism for chromosomes X, 9, and 15. In the case of AT, we observed a global expression of aneuploidy in up to 20-50% of cortex and cerebellum neurons. In addition, a local instability of chromosome 14 was revealed in the degenerating cerebellum in the form of breaks in the 14q12 region. In the case of AD, a tenfold increase was observed in the level ofaneuploidy for chromosome 21 in brain sections subjected to neurodegeneration. These data indicate that mosaic genomic instability in nerve cells is one of the mechanism of neurodegenerative and mental diseases.

[Chromosomal mosaicism in spontaneous abortions: analysis of 650 cases].

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase mFISH and DNAprobes for chromosomes 1,9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (approximately 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.

[Cytogenetic, molecular cytogenetic, clinical and genealogical study of mothers of children with autism: a search for family genetic markers of autistic disorders].

Using modern cytogenetic and molecular cytogenetic techniques towards the study of human chromosomes, an analysis of chromosomal abnormalities/chromosomal variations as well as clinical and genealogical data in mothers of children with autism has been performed. It has been shown that mothers of autistic children exhibit an increased incidence of chromosomal abnormalities (mainly mosaic forms involving chromosome X) and an increased occurrence of chromosomal variations compared to controls. The analysis of genotype-phenotype correlations revealed the increase in the frequency of cognitive disturbances and spontaneous abortions in mothers of children with autism as well as the higher frequency of mental retardation, early death and reproductive problems in the pedigrees. The high frequency of congenital malformations in the pedigrees of mothers with chromosomal variations was observed as well. Taking into account the data obtained, we have concluded that cytogenetic and molecular cytogenetic studies of mothers of children with autism are obligatory for detection of possible genetic causes of autism and genetic counseling of families with children affected with autistic disorders.

[Analysis of germ cell populations in ejaculate of men infected with herpes simplex virus].

Cytological and molecular genetics methods were used to study sperm from patients with sperm infected with herpes simplex virus (HSV) as indicated by virological and immunocytochemical tests. The following methods were used: (1) sperm analysis to evaluate the morphology and functional properties of sperm; (2) fluorescence in situ hybridization (FISH) with DNA probes specific for chromosomes 1, X, and Y to evaluate nondisjunction frequencies of these chromosomes in sperm; and (3) quantitative analysis of immature germ cells in the ejaculate to identify spermatogenic abnormalities. The total sperm count and the count of sperm with normal motility proved similar to the norm. FISH analysis demonstrated no difference in the nondisjunction frequency of chromosomes 1, X, and Y between infertile patients with HSV-infected sperm and fertile donors. Comparative quantitative analysis of immature germ cells from the ejaculate has demonstrated a significant and considerable (threefold) increase in the number of spermatocytes I at the prepachytene stages of prophase I (preleptotene, leptotene, and zygotene) in HSV patients compared to normal donors. At the same time, HSV patients demonstrated a significant decrease in the number of spermatocytes I, a decrease in the proportion of spermatocytes II and spermatids, and a twofold increase in the number of unidentifiable immature germ cells. The data obtained indicate a partial spermatogenic arrest at the early stages of meiotic prophase I in HSV patients, which prompts further research into the cellular mechanisms of abnormal spermatogenesis after viral infection in humans.

[Variations of heterochromatic chromosomal regions and chromosome abnormalities in children with autism: identification of genetic markers in autistic spectrum disorders].

In the present study, the cytogenetic and molecular cytogenetic analysis of 90 children with autism and their mothers (18 subjects) was carried out. Chromosome fragility and abnormalities were found in four cases: mos 47,XXX[98]/ 46,XX[2]; 46,XY,r(22)(p11q13); 46,XY,inv(2)(p11.2q13),16qh-; 46Y,fra(X)(q27.3)16qh-. Using C-banding and quantitative fluorescent in situ hybridization (FISH), the significantly increased incidence of heterochromatic region variation was shown in autism as compared to the controls (48 and 16%, respectively). Pericentric 9phqh inversion was not characteristic of the patients with autism whereas heterochromatic variations 1phqh, 9qh+ and 16qh- were more frequent in autism (p<0,05). Basing on the data obtained, a possible role of position effect in autism pathogenesis as well as a potential of heterochromatic region variation analysis for the search of biological markers of autistic spectrum disorders are discussed.

[Epigenetic study of Rett's syndrome as an adequate model for autistic disorders].

Rett's syndrome (RTT) is a severe hereditary disorder of the nervous system. MECP2 gene mutations are considered as a primary cause of the disease. In the present study, we have found MECP2 mutations in 33 (84.6%) out of 39 RTT females. We have also studied X-inactivation patterns in 70 girls with RTT. A frequency of skewed X-inactivation was 37% (26 patients), being significantly higher (p < 0.001) than that in the controls. The investigation of inactivated X chromosome origin revealed that about 33% pairs had preferentially the inactivated maternal X chromosome. An abnormal type of chromosome X inactivation was observed in all RTT females. Thus, we conclude that skewed X-inactivation may be considered as a common feature of RTT. There is unambiguous evidence that epigenetic alterations in RTT are associated with MECP2 mutations. MeCP2 protein also appears to be involved in transcriptional regulation of chromosome X genes. RTT in females without MECP2 mutations is related to the epigenetic alterations. We suggest X chromosome inactivation study in RTT females and their mothers to be informative for investigation of genetic processes in RTT girls, even in case MECP2 mutations have not been found. RTT could be considered as an appropriate model for studying epigenetic abnormalities in relation to autistic spectrum disorders.

[Diagnosis of numerical chromosomal aberrations in the cells of spontaneous abortions by multicolor fluorescence in situ hybridization (MFISH)].

According to different estimates, as high as 15-20% of all the pregnancies result in spontaneous abortions (SA) at different gestational periods. Identification of abnormalities leading to SA is of great importance for practical medicine, mainly for medical genetic counseling of married couples with impaired reproductive function. The diagnosis of chromosomal aberrations on the basis of SA materials is known to have a number of methodological difficulties. The present paper deals with the identification of numerical anomalies in the SA material by multicolor fluorescence in situ hybridization (MFISH). This technique using an original collection of DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y was applied to the study of chromosomal aberrations in 224 spontaneous abortion specimens. Numerical chromosomal aberrations were found in 122 (54.5%) cases. The cells of all the studied specimens exhibited aneuploidy of chromosome X in 17% cases; chromosome 16 in 12%, chromosomes 13/21 in 5.8%, chromosomes 14/22 in 4.9%, chromosomes 9 and 18 in 1.3% (each), chromosome 15 in 0.9%, chromosome 1 in 0.45%. Polyploidy was detected in 13.3% of cases; concomitant abnormalities were found in 7 cases. Analysis of the findings has led to the conclusion that MFISH can be successfully used in the diagnosis of numerical chromosomal aberrations of CA cells.

[Quantitative analysis of fluorescence in situ hybridization (FISH) signals for molecular cytogenetic diagnosis].

Molecular cytogenetic diagnosis of chromosomal aberrations, by using fluorescence in situ hybridization (FISH) requires the introduction of quantitative approaches to estimating hybridization signals. Specifically, this estimation is required for the differentiation of monosomy and for the superposition of the signals in the diagnosis of mosaic forms of chromosomal syndromes, as well for the determination of the parental origin of homologous chromosomes. The present paper proposes an approach to rapidly estimating FISH signals, based on image capture by a CCD digital chamber and a fluorescence microscope, and to measuring the intensity of hybridization signals. This approach has been shown to be highly effective in differentiating the parent origin of chromosomes, by applying original chromosome-specific DNA tests. It has been successfully used in the study of numerical chromosomal aberrations in various biological materials. By taking into account the findings and as well as earlier described quantitative FISH analyses applied to basic studies, the authors discuss whether the quantitative FISH analyses can be used for the molecular cytogenetic diagnosis of hereditary diseases.

[Molecular cytogenetic study of Robertsonian translocation 13;14 and Down syndrome in a 3-year-old infant]. / Molekuliano-tsitogeneticheskoe issledovanie Robertsonovskoi translokatsii 13;14 i sindroma Dauna u rebenka 3 let.

We describe here a rare case of Robertsonian translocation 13;14 of maternal origin combined with regular trisomy 21 (46,XX,der(13;14)(q10;q10)mat,+21) with Down syndrome phenotype. Molecular cytogenetic studies allowed us to determine the maternal origin of additional chromosome 21 and the non-disjunction of chromosome 21 to occur in meiosis I. On the basis of data obtained we discuss the possible involvement of structural alterations of chromosomes 13 and 14 in the chromosome 21 non-disjunction.

[X chromosome inactivation pattern in elderly women over 70 years of age]. / Osobennosti inaktivatsii khromosomy X u pozhilykh zhenshchin starshe 70 let.

In the present study we have analyzed X chromosome inactivation patterns in 40 women aged from 74 to 85 years (mean age 78 years). The control group was 36 women (mean age 30 years). The most common AR-assay was used to determine X-inactivation patterns (the study of methylation patterns of HpaII site in human androgen receptor gene (HUMARA) by quantative PCR). The age dependence of X-inactivation was not observed. We have detected skewed X-inactivation in three women among 40 (7.5%) elderly women comparing to two women among 36 (5.5%) women from control group. The difference was not found to be statistically significant. We made a suggestion that higher incidence of skewed X-inactivation in elderly women revealed by previous studies could occur due to some experimental ambiguities as heterogeneity of the group studied; inclusion of women having relatives with genetic abnormalities associated with skewed X-inactivation patterns; the difference of X chromosome inactivation skewing determination. We conclude that present study does not show X chromosome inactivation to be age dependent.

[Genotype-phenotype correlations in Rett syndrome: the study of Russian cohort of patients]. / Kliniko-geneticheskie korreliatsii pri sindrome Retta: izuchenie rossiiskoi kogorty bol'nykh.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein 2 gene (MECP2). We carried out a mutations analysis in Russian cohort of patients with RTT. MECP2 mutations were found in 23 of 28 RTT girls and one boy (82%). Thirteen different types of mutations have been identified: 6 nonsense, 5 missense and 2 deletions in MECP2 gene. In typical RTT form, most frequent mutations were R255X (5 cases) and T158M (4 cases). A boy with classical clinical picture of RTT had R270X mutation. Skewed inactivation of chromosome x has been found in 2 of 27 RTT girls with classical RTT form and "forme fruste". The data obtained imply possible correlations between genotype and phenotype in RTT.